Our group has recently developed a novel fluorescence method that allows the measurement of the transverse location (depth) of specific sites within a membrane relative at the angstrom level of resolution. The approach, which we have named the parallax analysis, involves fluorescence quenching measurements using phospholipids carrying spin- labeled quenchers. We derived simple algebraic expressions that allow calculation of fluorophore depth from such experiments. The aim of this project is to further develop and apply the method to solve questions of membrane structure. Specifically: 1) The method will be applied to a wide range of membrane probes to determine the chemical/structural factors that control depth of molecules in membranes. By correlation of depth with polarity, charge, number of heteroatoms, hydrogen bonding, and other structural features it should be possible to identify which properties influence depth most strongly. 2) The applicability of the method will be extended to the extremities of the polar headgroup and adjacent aqueous solution by the use of a spin-label attached to the polar headgroup of a lipid. Another goal is to extend the method to distinguish which half of a bilayer a group is found in, an important parameter for membrane-inserted proteins. this will be done by using ascorbate reduction of spin-labels in half of the membrane to restrict quenching to a single leaflet of the bilayer. 3) In collaborative studies the method will be used to analyze the topography of membrane proteins. To accomplish this the location of single fluorescent Trp residues substituted into the alpha-helical membrane protein lactose permease will be determined. Similar studies will be undertaken with the pore-forming beta sheet alpha-toxin of S. aureus. In this case, single fluorescent sites will be created by labeling of mutants containing single Cys residues. Together these studies should establish the method as a practical approach to questions of membrane structure and function.